Golgi export of the Kir2.1 channel is driven by a trafficking signal located within its tertiary structure.

Mechanisms that are responsible for sorting newly synthesized proteins for traffic to the cell surface from the Golgi are poorly understood. Here, we show that the potassium channel Kir2.1, mutations in which are associated with Andersen-Tawil syndrome, is selected as cargo into Golgi export carriers in an unusual signal-dependent manner. Unlike conventional trafficking signals, which are typically comprised of short linear peptide sequences, Golgi exit of Kir2.1 is dictated by residues that are embedded within the confluence of two separate domains. This signal patch forms a recognition site for interaction with the AP1 adaptor complex, thereby marking Kir2.1 for incorporation into clathrin-coated vesicles at the trans-Golgi. The identification of a trafficking signal in the tertiary structure of Kir2.1 reveals a quality control step that couples protein conformation to Golgi export and provides molecular insight into how mutations in Kir2.1 arrest the channels at the Golgi.

Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger.

The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 µM) and CBD2 (K(d) = 18.4 ± 6 µM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.

Chain-reaction Ca(2+) signaling in the heart.

Mutations in Ca(2+) -handling proteins in the heart have been linked to exercise-induced sudden cardiac death. The best characterized of these have been mutations in the cardiac Ca(2+) release channel known as the ryanodine receptor type 2 (RyR2). RyR2 mutations cause "leaky" channels, resulting in diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR) that can trigger fatal cardiac arrhythmias during stress. In this issue of the JCI, Song et al. show that mutations in the SR Ca(2+)-binding protein calsequestrin 2 (CASQ2) in mice result not only in reduced CASQ2 expression but also in a surprising, compensatory elevation in expression of both the Ca(2+)-binding protein calreticulin and RyR2, culminating in premature Ca(2+) release from cardiac myocytes and stress-induced arrhythmia (see the related article beginning on page 1814). In the context of these findings and other recent reports studying CASQ2 mutations, we discuss how CASQ2 influences the properties of Ca(2+)-dependent regulation of RyR2 and how this contributes to cardiac arrhythmogenesis.

Nuclear export of misfolded SOD1 mediated by a normally buried NES-like sequence reduces proteotoxicity in the nucleus.

Over 170 different mutations in the gene encoding SOD1 all cause amyotrophic lateral sclerosis (ALS). Available studies have been primarily focused on the mechanisms underlying mutant SOD1 cytotoxicity. How cells defend against the cytotoxicity remains largely unknown. Here, we show that misfolding of ALS-linked SOD1 mutants and wild-type (wt) SOD1 exposes a normally buried nuclear export signal (NES)-like sequence. The nuclear export carrier protein CRM1 recognizes this NES-like sequence and exports misfolded SOD1 to the cytoplasm. Antibodies against the NES-like sequence recognize misfolded SOD1, but not native wt SOD1 both in vitro and in vivo. Disruption of the NES consensus sequence relocalizes mutant SOD1 to the nucleus, resulting in higher toxicity in cells, and severer impairments in locomotion, egg-laying, and survival in Caenorhabditis elegans. Our data suggest that SOD1 mutants are removed from the nucleus by CRM1 as a defense mechanism against proteotoxicity of misfolded SOD1 in the nucleus.

Synthesis and Characterization of 8-O-Carboxymethylpyranine (CM-Pyranine) as a Bright, Violet-Emitting, Fluid-Phase Fluorescent Marker in Cell Biology.

To avoid spectral interference with common fluorophores in multicolor fluorescence microscopy, a fluid-phase tracer with excitation and emission in the violet end of the visible spectrum is desirable. CM-pyranine is easily synthesized and purified. Its excitation and emission maxima at 401.5 nm and 428.5 nm, respectively, are well suited for excitation by 405-nm diode lasers now commonly available on laser-scanning microscopes. High fluorescence quantum efficiency (Q = 0.96) and strong light absorption (ε405 > 25,000 M-1cm-1) together make CM-pyranine the brightest violet aqueous tracer. The fluorescence spectrum of CM-pyranine is invariant above pH 4, which makes it a good fluid-phase marker in all cellular compartments. CM-pyranine is very photostable, is retained for long periods by cells, does not self-quench, and has negligible excimer emission. The sum of its properties make CM-pyranine an ideal fluorescent tracer. The use of CM-pyranine as a fluid-phase marker is demonstrated by multicolor confocal microscopy of cells that are also labeled with lipid and nuclear markers that have green and red fluorescence emission, respectively.

A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes.

The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.

Deglycosylation of the beta1-subunit of the BK channel changes its biophysical properties.

Large-conductance Ca(2+)-activated potassium (BK) channels are composed of pore-forming alpha-subunits and auxiliary beta-subunits. The alpha-subunits are widely expressed in many cell types, whereas the beta-subunits are more tissue specific and influence diverse aspects of channel function. In the current study, we identified the presence of the smooth muscle-specific beta1-subunit in murine colonic tissue using Western blotting. The native beta1-subunits migrated in SDS-PAGE as two molecular mass bands. Enzymatic removal of N-linked glycosylations from the beta1-subunit resulted in a single band that migrated at a lower molecular mass than the native beta1-subunit bands, suggesting that the native beta1-subunit exists in either a core glycosylated or highly glycosylated form. We investigated the functional consequence of deglycosylating the beta1-subunit during inside-out single-channel recordings. During inside-out single-channel recordings, with N-glycosidase F in the pipette solution, the open probability (P(o)) and mean open time of BK channels increased in a time-dependent manner. Deglycosylation of BK channels did not affect the conductance but shifted the steady-state voltage of activation toward more positive potentials without affecting slope when Ca(2+) concentration was <1 microM. Treatment of myocytes lacking the beta1-subunits of the BK channel with N-glycosidase F had no effect. These data suggest that glycosylations on the beta1-subunit in smooth muscle cells can modify the biophysical properties of BK channels.

VIP and PACAP regulate localized Ca2+ transients via cAMP-dependent mechanism.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been suggested as participants in enteric inhibitory neural regulation of gastrointestinal motility. These peptides cause a variety of postjunctional responses including membrane hyperpolarization and inhibition of contraction. Neuropeptides released from enteric motor neurons can elicit responses by direct stimulation of smooth muscle cells as opposed to other transmitters that rely on synapses between motor nerve terminals and interstitial cells of Cajal. Therefore, we studied the responses of murine colonic smooth muscle cells to VIP and PACAP(1-38) with confocal microscopy and patch-clamp technique. Localized Ca2+ transients (Ca2+ puffs) were observed in colonic myocytes, and these events coupled to spontaneous transient outward currents (STOCs). VIP and PACAP increased Ca2+ transients and STOC frequency and amplitude. Application of dibutyryl cAMP had similar effects. The adenylyl cyclase blocker MDL-12,330A alone did not affect spontaneous Ca2+ puffs and STOCs but prevented responses to VIP. Disruption of A-kinase-anchoring protein (AKAP) associations by application of AKAP St-Ht31 inhibitory peptide had effects similar to those of MDL-12,330A. Inhibition of ryanodine receptor channels did not block spontaneous Ca2+ puffs and STOCs but prevented the effects of dibutyryl cAMP. These findings suggest that regulation of Ca2+ transients (which couple to activation of STOCs) may contribute to the inhibitory effects of VIP and PACAP. Regulation of Ca2+ transients by VIP and PACAP occurs via adenylyl cyclase, increased synthesis of cAMP, and PKA-dependent regulation of ryanodine receptor channels.

Beta 1-subunits are required for regulation of coupling between Ca2+ transients and Ca2+-activated K+ (BK) channels by protein kinase C.

Colonic myocytes have spontaneous, localized, Ins (1,4,5) trisphosphate (IP3) receptor-dependent Ca2+ transients that couple to the activation of Ca2+-dependent K+ channels and spontaneous transient outward currents (STOCs). We previously reported that the coupling strength between spontaneous Ca2+ transients and large conductance Ca2+ activated K+ (BK) channels is regulated by Ca2+ influx through nonselective cation channels and activation of protein kinase C (PKC). Here, we used confocal microscopy and the patch-clamp technique to further investigate the coupling between localized Ca2+ transients and STOCs in colonic myocytes from animals lacking the regulatory beta1-subunit of BK channels. Myocytes from beta1-knockout (beta1-/-) animals loaded with fluo 4 showed typical localized Ca2+ transients, but the STOCs coupled to these events were of abnormally low amplitude. Reduction in external Ca2+ or application of inhibitors of nonselective cation channels (SKF-96365) caused no significant change in the amplitude or frequency of STOCs. Likewise, an inhibitor of PKC, GF 109203X, had no significant effect on STOCs. Single-channel recording from BK channels showed that application of an activator (PMA) and an inhibitor (GF 109203X) of PKC did not affect BK channel openings in myocytes of beta1-/- mice. These data show that PKC-dependent regulation of coupling strength between Ca2+ transients and STOCs in colonic myocytes depends upon the interaction between alpha- and beta1-subunits.